Shu Hui Juan

Adiponectin-Mediated Heme Oxygenase-1 Induction Protects Against Iron-Induced Liver Injury via a PPARα-Dependent Mechanism

Protective effects of adiponectin (APN; an adipocytokine) were shown against various oxidative challenges; however, its therapeutic implications and the mechanisms underlying hepatic iron overload remain unclear. Herein, we show that the deleterious effects of iron dextran on liver function and iron deposition were significantly reversed by adiponectin gene therapy, which was accompanied by AMP-activated protein kinase (AMPK) phosphorylation and heme oxygenase (HO)-1 induction. Furthermore, AMPK-mediated peroxisome proliferator-activated receptor-α (PPARα) activation by APN was ascribable to HO-1 induction. Additionally, we revealed direct transcriptional regulation of HO-1 by the binding of PPARα to a PPAR-responsive element (PPRE) by various experimental assessments. Interestingly, overexpression of HO-1 in hepatocytes mimicked the protective effect of APN in attenuating iron-mediated injury, whereas it was abolished by SnPP and small interfering HO-1. Furthermore, bilirubin, the end-product of the HO-1 reaction, but not CO, protected hepatocytes from iron dextran-mediated caspase activation. Herein, we demonstrate a novel functional PPRE in the promoter regions of HO-1, and APN-mediated HO-1 induction elicited an antiapoptotic effect and a decrease in iron deposition in hepatocytes subjected to iron challenge.

Molecular mechanisms of the antiproliferative effect of beraprost, a prostacyclin agonist, in murine vascular smooth muscle cells

Prostacyclin (PGI2) has been shown to inhibit proliferation in vascular smooth muscle cells. To clarify the underlying molecular mechanism, we investigated the vasoprotection of beraprost (a PGI2 agonist) both in vivo and in vitro. Beraprost eliminated increases in proliferation of rat aortic smooth muscle cells (RASMCs) by 12‐O‐tetradecanoylphorbol 13‐acetate, and enhanced the peroxisome proliferator‐activated receptor‐delta (PPARδ) and inducible nitric oxide synthetase (iNOS) expressions, which were associated with the antiproliferative action of beraprost according to inhibition experiments by [3H]thymidine incorporation. Additionally, elimination of iNOS activity by PPARδ antagonists suggested that iNOS is the downstream target of PPARδ. Furthermore, beraprost increased both consensus PPARδ‐responsive element (PPRE)‐driven luciferase activity and the binding activity of the PPARδ to the putative PPRE in the iNOS promoter; nevertheless, it was abolished by PPARδ antagonists. Deletion of PPRE (−1,349/−1,330) in the iNOS promoter region (−1,359/+2) strongly reduced promoter‐driven activity, representing a novel mechanism of iNOS induction by beraprost. Consistent with this, PPARδ and the concomitant iNOS induction by beraprost were also evident in vivo. Beraprost‐mediated protection in a murine model of balloon angioplasty was significantly attenuated by 13S‐HODE, a PPARδ antagonist. Taken together, the results suggest that the causal relationship between PPARδ and iNOS contributes to the vasoprotective action of beraprost in RASMCs. J. Cell. Physiol. 214: 434–441, 2008.

Mechanism of Heme Oxygenase-1 Gene Induction by Quercetin in Rat Aortic Smooth Muscle Cells

We previously reported that adenovirus-mediated gene transfer of heme oxygenase-1 (HO-1) inhibits the development of atherosclerosis in apolipoprotein E-deficient mice. This finding implies that HO-1 induction is beneficial for protecting blood vessels. We also found that quercetin, a common polyphenolic compound in foods of plant origin, induces HO-1 expression in RAW264.7 cells. This study was aimed at examining the potency of quercetin as a HO-1 inducer and its regulation in rat aortic smooth muscle cells (RASMCs). We showed that quercetin-induced HO-1 production was in time- and dose-dependent fashions, and that this regulation occurred at both transcription and translation levels. Quercetin increased p38 mitogen-activated protein kinase (p38MAPK), but inhibited extracellular signal-regulated kinase in RASMCs. The level of quercetin-induced HO-1 expression was attenuated by SB202190 (a p38MAPK inhibitor). Taken together from the data in this study, we suggest that quercetin induced HO-1 expression, at least in part, through p38MAPK.

PPARδ-mediated p21/p27 induction via increased CREB-binding protein nuclear translocation in beraprost-induced antiproliferation of murine aortic smooth muscle cells

We previously showed that an increase in the peroxisome proliferator-activated receptor-delta (PPARdelta), together with subsequent induction of inducible nitric oxide synthase (iNOS) by beraprost (BPS), inhibits aortic smooth muscle cell proliferation. Herein, we delineated the mechanisms of the antiproliferative effects of BPS through the induction of p21/p27. BPS concentration dependently induced the p21/p27 promoter- and consensus cAMP-responsive element (CRE)-driven luciferase activities, which were significantly suppressed by blocking PPARdelta activation. Surprisingly, other than altering the CRE-binding protein (CREB), BPS-mediated PPARdelta activation increased nuclear localization of the CREB-binding protein (CBP), a coactivator, which was further confirmed by chromatin immunoprecipitation. Furthermore, novel functional PPAR-responsive elements (PPREs) next to CREs in the rat p21/p27 promoter regions were identified, where PPARdelta interacted with CREB through CBP recruitment. BPS-mediated suppression of restenosis in mice with angioplasty was associated with p21/p27 induction. Herein, we demonstrate for the first time that BPS-mediated PPARdelta activation enhances transcriptional activation of p21/p27 by increasing CBP nuclear translocation, which contributes to the vasoprotective action of BPS.

Aryl-hydrocarbon receptor-dependent alteration of FAK/RhoA in the inhibition of HUVEC motility by 3-methylcholanthrene

We previously demonstrated the antiproliferative and antiangiogenic effects of 3-methylcholanthrene (3MC), an aryl-hydrocarbon receptor (AhR) agonist, in human umbilical vascular endothelial cells (HUVECs). Herein, we unraveled its molecular mechanisms in inhibiting HUVEC motility. 3MC down-regulated FAK, but up-regulated RhoA, which was rescued by AhR knockdown. It led us to identify novel AhR binding sites in the FAK/RhoA promoters. Additionally, 3MC increased RhoA activity via suppression of a negative feedback pathway of FAK/p190RhoGAP. With an increase in membrane-bound RhoA, subsequent stress fiber and focal adhesion complex formation was observed in 3MC-treated cells, and this was reversed by a RhoA inhibitor and AhR antagonists. Notably, these compounds significantly reversed 3MC-mediated anti-migration in a transwell assay. The in vitro findings were further confirmed using an animal model of Matrigel formation in Balb/c mice. Collectively, AhR’s genomic regulation of FAK/RhoA, together with RhoA activation, is ascribable to the anti-migration effect of 3MC in HUVECs.

Potential Mechanism of Blood Vessel Protection by Resveratrol, a Component of Red Wine

Abstract: Resveratrol‐mediated heme oxgenase‐1 (HO‐1) induction has been shown to occur in primary neuronal cultures and has been implicated as having potential neuroprotective action. Further, antioxidant properties of resveratrol have been reported to protect against coronary heart disease. We attempted to examine the HO‐1 inducing potency of resveratrol and the regulatory mechanism of its induction in rat aortic smooth muscle cells (RASMC). We showed that resveratrol‐induced HO‐1 expression was concentration‐ and time‐dependent. The level of HO‐1 expression and its promoter activity mediated by resveratrol was attenuated by nuclear factor‐kappa B (NF‐κB) inhibitors, but not by mitogen‐activated protein kinase (MAPK) inhibitors. Deletion of NF‐κB binding sites in the promoter region strongly reduced luciferase activity. Collectively, we suggest that resveratrol‐mediated HO‐1 expression occurs, at least in part, through the NF‐κB pathway.

Mechanisms of Adiponectin-Mediated COX-2 Induction and Protection Against Iron Injury in Mouse Hepatocytes

Adiponectin (APN)‐mediated cyclooxygenase (COX)‐2 induction is known to have various protective effects on cardiovascular diseases. However, the molecular mechanisms of APN‐mediated COX‐2 induction and its protection against iron‐mediated injury in hepatocytes are still unclear. Herein, we show that AMP‐mediated peroxisome proliferator‐activated receptor (PPAR)α activation was attributable to COX‐2 induction by APN, which was further confirmed by identifying novel functional PPAR responsive elements (PPREs) in the mouse COX‐2 promoter region. Prostaglandin (PG)I2 and PGE2, metabolites of COX‐2, time‐dependently increased in hepatocytes treated with APN. Interestingly, beraprost and misoprostol, respective agonists for PGI2 and PGE2, mimicked the protective effects of APN in iron‐mediated inflammation in hepatocytes. The iron dextran‐activated nuclear factor (NF)‐κB pathway was correlated with the increased production of inflammatory cytokines including tumor necrosis factor‐α, intercellular adhesion molecule‐1, and monocyte chemotactic protein‐1. This was eliminated by administration of APN, whereas blockage of PPARα activation, an upstream regulator of COX‐2 induction by APN, and COX‐2 activation reversed the anti‐inflammatory effect of APN, suggesting the crucial role of COX‐2 in this event. Herein, we demonstrate that APN‐mediated COX‐2 induction through a PPARα‐dependent mechanism, and COX‐2 exerted an anti‐inflammatory effect of APN in hepatocytes subjected to iron challenge. J. Cell. Physiol. 224: 837–847, 2010.

3-Methylcholanthrene, an AhR Agonist, Caused Cell-Cycle Arrest by Histone Deacetylation through a RhoA-Dependent Recruitment of HDAC1 and pRb2 to E2F1 Complex

We previously showed that treating vascular endothelial cells with 3-methylcholanthrene (3MC) caused cell-cycle arrest in the Go/G1 phase; this resulted from the induction of p21 and p27 and a decreased level and activity of the cyclin-dependent kinase, Cdk2. We further investigated the molecular mechanisms that modulate cell-cycle regulatory proteins through the aryl-hydrocarbon receptor (AhR)/Ras homolog gene family, member A (RhoA) dependent epigenetic modification of histone. AhR/RhoA activation mediated by 3MC was essential for the upregulation of retinoblastoma 2 (pRb2) and histone deacetylase 1 (HDAC1), whereas their nuclear translocation was primarily modulated by RhoA activation. The combination of increased phosphatase and tensin homolog (PTEN) activity and decreased phosphatidylinositide 3-kinase (PI3K) activation by 3MC led to the inactivation of the Ras-cRaf pathway, which contributed to pRb2 hypophosphorylation. Increased HDAC1/pRb2 recruitment to the E2F1 complex decreased E2F1-transactivational activity and H3/H4 deacetylation, resulting in the downregulation of cell-cycle regulatory proteins (Cdk2/4 and Cyclin D3/E). Co-immunoprecipitation and electrophoretic mobility shift assay (EMSA) results showed that simvastatin prevented the 3MC-increased binding activities of E2F1 proteins in their promoter regions. Additionally, RhoA inhibitors (statins) reversed the effect of 3MC in inhibiting DNA synthesis by decreasing the nuclear translocation of pRb2/HDAC1, leading to a recovery of the levels of cell-cycle regulatory proteins. In summary, 3MC decreased cell proliferation by the epigenetic modification of histone through an AhR/RhoA-dependent mechanism that can be rescued by statins.

L-carnitine protects against carboplatin-mediated renal injury: AMPK- And PPARα-dependent inactivation of NFAT3

We have previously shown that carboplatin induces inflammation and apoptosis in renal tubular cells (RTCs) through the activation of the nuclear factor of activated T cells-3 (NFAT3) protein by reactive oxygen species (ROS), and that the ROS-mediated activation of NFAT3 is prevented by N-acetyl cysteine and heme oxygenase-1 treatment. In the current study, we investigated the underlying molecular mechanisms of the protective effect of L-carnitine on carboplatin-mediated renal injury. Balb/c mice and RTCs were used as model systems. Carboplatin-induced apoptosis in RTCs was examined using terminal-deoxynucleotidyl-transferase-mediated dUTP nick end labeling. We evaluated the effects of the overexpression of the peroxisome-proliferator-activated receptor alpha (PPARα) protein, the knockdown of PPARα gene, and the blockade of AMPK activation and PPARα to investigate the underlying mechanisms of the protective effect of L-carnitine on carboplatin-mediated renal injury. Carboplatin reduced the nuclear translocation, phosphorylation, and peroxisome proliferator responsive element transactivational activity of PPARα. These carboplatin-mediated effects were prevented by L-carnitine through a mechanism dependent on AMPK phosphorylation and subsequent PPARα activation. The activation of PPARα induced cyclooxygenase 2 (COX-2) and prostacyclin (PGI2) synthase expression that formed a positive feedback loop to further activate PPARα. The coimmunoprecipitation of the nuclear factor (NF) κB proteins increased following the induction of PPARα by L-carnitine, which reduced NFκB transactivational activity and cytokine expression. The in vivo study showed that the inactivation of AMPK suppressed the protective effect of L-carnitine in carboplatin-treated mice, indicating that AMPK phosphorylation is required for PPARα activation in the L-carnitine-mediated protection of RTC apoptosis caused by carboplatin. The results of our study provide molecular evidence that L-carnitine prevents carboplatin-mediated apoptosis through AMPK-mediated PPARα activation.

Ectopic overexpression of haem oxygenase-1 protects kidneys from carboplatin-mediated apoptosis

BACKGROUND AND PURPOSE We previously reported that the activation of the nuclear factor of activated T‐lymphocyte‐3 (NFAT3) by carboplatin leads to renal apoptosis as a result of oxidative stress, which is reversed by N‐acetylcysteine. Herein, we extend our previous work to provide evidence of the molecular mechanisms of haem oxygenase (HO)‐1 in protecting against injury.